Download Chorioamnionitis induced by intraamniotic lipopolysaccharide

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Chorioamnionitis induced by intraamniotic lipopolysaccharide
resulted in an interval-dependent increase in central
nervous system injury in the fetal sheep
A. W. Danilo Gavilanes, MD, PhD; Eveline Strackx, MSc; Boris W. Kramer, MD, PhD; Markus Gantert, MD;
Daniël Van den Hove, PhD; Hellen Steinbusch; Yves Garnier, MD, PhD; Erwin Cornips, MD;
Harry Steinbusch, PhD; Luc Zimmermann, MD, PhD; Johan Vles, MD, PhD
OBJECTIVE: We quantified the impact of chorioamnionitis on both the
white and gray matter structures of the preterm ovine central nervous
system (CNS).
STUDY DESIGN: The CNS was studied at 125 days of gestation, either
2 or 14 days after the intraamniotic administration of 10 mg of lipopolysaccharide (LPS) (Escherichia coli) or saline. Apoptotic cells and
cell types were analyzed in the brain, cerebellum, and spinal cord using flow cytometry.
RESULTS: Apoptosis and microglial activation increased in all re-
gions with prolonged exposure to LPS-induced chorioamnionitis.
Astrocytes were increased in the brain and cerebellum of LPS-exposed fetuses but not in the spinal cord. Mature oligodendrocytes
decreased in the cerebral and cerebellar white matter, the cerebral
cortex, caudate putamen, and hippocampus 14 days after LPS.
Neurons in the cerebral cortex, hippocampus, and substantia nigra
were reduced 14 days after LPS.
CONCLUSION: Fetal inflammation globally but differentially affected
the CNS depending on the maturational stage of the brain region.
Key words: central nervous system, chorioamnionitis, fetal
inflammation, lipopolysaccharide, oligodendrocytes
Cite this article as: Gavilanes AWD, Strackx E, Kramer BW, et al. Chorioamnionitis induced by intraamniotic lipopolysaccharide resulted in an interval-dependent
increase in central nervous system injury in the fetal sheep. Am J Obstet Gynecol 2009;200:437.e1-437.e8.
C
horioamnionitis (CA) and the corresponding fetal inflammatory response are both inversely related to gestational age in preterm infants,1-3 affecting
mainly the lungs and the central nervous
system (CNS).4-7 The predominantly
studied CNS pathology is cerebral periventricular white matter (WM) disease
(WMD), which results in permanent
structural brain damage and severe longlasting neurodevelopmental impairment,
such as periventricular leukomalacia.8,9
Cortical and subcortical gray matter (GM)
and cerebellum are affected as well, contributing to a complex long-term neurologic morbidity.10,11
This study aimed to overcome 2 general
drawbacks from the most commonly used
experimental paradigms. First, lipopolysaccharide (LPS) is usually given intravenously (IV) to induce fetal inflammation.
In addition to fetal inflammation, how-
Received May 26, 2008; revised July 20, 2008; accepted Dec. 4, 2008.
ever, this approach also induces important
superimposed hypoxia ischemia.12 Second, little attention has been paid to other
CNS regions besides the cerebral WM. To
overcome the first drawback, we studied
the sheep CNS pathology after CA by LPS
administration into the amniotic fluid.
This intraamniotic administration avoids
the hemodynamic changes and the secondary postasphyctic encephalopathy induced by fetal IV LPS.13,14 In addition, this
experimental time window corresponds to
approximately 28 weeks of human CNS
maturation, which is the most vulnerable
period for the human brain to develop
WMD.15-17 To overcome the second
drawback, we studied the regional impact
on the developing CNS in different regions
of the WM and GM using flow cytometry.
We hypothesize both a global CNS impact
and a time-related effect after LPS-induced
CA.
Reprints: A. W. Danilo Gavilanes, MD, Department of Pediatrics, Division of Neonatology,
Maastricht University Medical Center, PO Box 5800, NL-6200 AZ Maastricht, The Netherlands.
[email protected].
M ATERIALS AND M ETHODS
From the Departments of Pediatrics–Neonatology (Drs Gavilanes, Kramer, and
Zimmermann and Ms Strackx), Child Neurology (Ms Strackx and Dr Vles), and
Neurosurgery (Dr Cornips), University Hospital Maastricht, in collaboration with the
Research Institute of Growth and Development (GROW), Maastricht, The Netherlands; the
Faculty of Health, Medicine and Life Sciences, School of Mental Health and Neuroscience,
Maastricht University, and European Graduate School of Neuroscience (EURON),
Maastricht, The Netherlands (Dr Van den Hove, and Ms Hellen Steinbusch, Mr Harry
Steinbusch, and Ms Strackx); and the Department of Obstetrics and Gynecology, University
of Köln, Köln, Germany (Drs Gantert and Garnier).
The first 2 authors contributed equally to this research.
0002-9378/$36.00 • © 2009 Mosby, Inc. All rights reserved. • doi: 10.1016/j.ajog.2008.12.003
Animals and surgical procedures
All experimental procedures were approved by our animal ethics board accord-
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FIGURE 1
Body and brain weights
Effect of fetal endotoxin exposure on body and
brain weight. A, No significant differences in
bodyweight between groups. B, Brain weight
was significantly lower in lipopolysaccharide
(LPS) 2-day (2D) and 14-day (14D) groups than
in control group. C, Endotoxin exposure did not
cause any changes in brain to body ratio among
3 groups. Data are represented as mean ⫹ SEM.
Black bars ⫽ control (n ⫽ 7); gray bars ⫽ LPS
2D (n ⫽ 5); and white bars ⫽ LPS 14D (n ⫽
6) groups. Multivariate analysis of variance ⫹
Bonferroni testing; *P ⬍ .05 and ***P ⬍ .001.
Gavilanes. CA induced by intraamniotic LPS resulted in an
interval-dependent increase in CNS injury in the fetal sheep.
Am J Obstet Gynecol 2009.
ing to Dutch government regulations.
Pregnant Texel ewes, bearing both singletons and twins, were housed outdoors.
Food and water were provided ad libitum.
LPS (10 mg dissolved in 2 mL sterile and
filtered saline) was injected intraamniotically (IA) under ultrasound guidance at
day 123 of gestation (n ⫽ 5) or at day 111 of
gestation (n ⫽ 6). Control saline injection
was given at either day 111 or 123 of gestation (n ⫽ 7).18 At day 125 of gestation,
pregnant ewes were anesthetized and all fetuses were delivered by cesarean section.
Fetuses were killed by a lethal injection of
pentobarbital. The brain was removed and
halved. One half was prepared for flow cytometric analysis.
437.e2
Tissue preparation
The cerebral cortex, caudate nucleus and
putamen (CPU), hippocampus, frontal
periventricular WM, hypothalamus, substantia nigra (SN), C1, T11, and L1 of the
spinal cord (SC), and cerebellar cortex and
WM were dissected and placed in ice-cold
plating medium consisting of Dulbecco’s
Modified Eagle’s medium supplemented
with 10% fetal bovine serum (FBS), penicillin/streptavidin, and glutamate. The tissue was mechanically disrupted using a
glass homogenizer. The suspension was
centrifuged at 1200 rpm at 4°C for 10 minutes, and the pellet was resuspended in 5
mL of plating medium. The crude cell suspension was then passed through a
100-␮m nylon cell strainer to remove large
cell clumps. The cells were counted using a
cell counting chamber and divided into
different microcentrifuge tubes (106 cells/
0.5 mL/tube). The viability of the cells was
monitored using trypan blue staining.
Flow cytometric analysis
AnnexinV/propidium iodide staining
Apoptotic and necrotic cells were detected with annexinV conjugated to
fluorescin (FITC) and propidium iodide
(PI) (AnnexinV-FITC Apoptosis Detection Kit; BD Biosciences Pharmingen,
Breda, The Netherlands). The cells were
washed twice with phosphate buffered
saline (PBS), followed by a wash in AnnexinV-binding buffer. Then, all samples (107 cells/100 ␮L) were incubated
with 5 ␮L of AnnexinV antibody and 5
␮L of PI for 15 minutes at room temperature (RT).
Extracellular staining (OX42)
OX42 was used to identify activated microglia. The cells were washed twice with
staining buffer (PBS ⫹ 2% FBS) by centrifugation at 1200 rpm at 4°C and incubated for 30 minutes at RT with the primary antibody: mouse antibovine
CD11b/c (clone OX42; AbD Serotec,
Huissen, The Netherlands), diluted
1:100 in staining buffer. The cells were
washed twice followed by incubation
with the secondary antibody: donkey antimouse alexa 488 diluted 1:100 in staining buffer. All samples were washed and
kept at 4°C in the dark.
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Intracellular staining (glial fibrillary
acidic protein, 2=,3=-cyclic nucleotide
3=-phosphodiesterase, and
antineurofilament 200-kd)
Antiglial fibrillary acidic protein antibodies were used to identify activated astrocytes. Anti-2=,3=-cyclic nucleotide 3=phosphodiesterase antibodies were used to
detect mature oligodendrocytes, whereas
antineurofilament 200-kd antibodies were
used to detect neurons. Cells were washed
twice with staining buffer by centrifugation at 1200 rpm at 4°C. After washing,
cells were fixed with 4% paraformaldehyde
in staining buffer (2% final concentration)
for 20 minutes at RT. Permeabilization was
done after 2 more washing steps with permeabilization buffer (0.05% saponin ⫹
2% FBS ⫹ PBS) for 10 minutes at RT. The
cell suspension was then incubated with
the primary antibody for 30 or 45 minutes
at RT. The antibodies were mouse antiovine 2=,3=-cyclic nucleotide phosphodiesterase (clone 11-5B; Sigma, Munich, Germany); monoclonal antibody 1:200 in
permeabilization buffer/rabbit antiovine
glial fibrillary acidic protein (Dako,
Glostrup, Denmark); and polyclonal antibody 1:200 in permeabilization buffer/
mouse antineurofilament 200-kd (Abcam,
Cambridge, UK); and monoclonal antibody 1:100 in permeabilization buffer. After 2 washing steps, the cell suspension was
incubated with the secondary antibody
(respectively donkey antimouse or donkey
antirabbit alexa 488 diluted 1:100 in permeabilization buffer) again for 30 minutes
at RT. All samples were washed 2 times and
kept at 4°C in the dark.
Flow cytometry
All samples were run on an FACScalibur
flow cytometry system (BD Biosciences,
San Jose, CA), equipped with an argon
ion laser (488 nm). Analysis was done
using the Cell Quest Pro software (BD
Biosciences). Forward and sideward
light angle scatters were collected from
all samples. Using those plots, samples
were gated (region 1; R1) to exclude cell
debris and cellular aggregates for further
analysis. For each marker, the mean fluorescence intensity and the percentage of
positive cells stained above background
were measured for a total of 10,000 cells
per sample within the gate (R1). The cut-
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FIGURE 2
Flow cytograms
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off was defined using control tissue negative for the different markers processed
and stained alongside the experimental
samples. The mean fluorescence intensity was corrected for autofluorescence
using the values from cells only incubated with secondary antibody.
Statistical analysis
Data were analyzed using a multivariate
analysis of variance (mean ⫾ SEM). Significant effects were analyzed by post hoc
Bonferroni corrections. The accepted
level of significance was P ⬍ .05. All calculations were done using software
(SPSS 12.0; SPSS, Inc, Chicago, IL).
Representative flow cytograms of control and
lipopolysaccharide (LPS) 14-day (14D) animals.
Flow cytograms of AnnexinV (AnnV) binding vs
propidium iodide (PI) uptake of A, control and
B, LPS 14D animals. Four populations of cells
are visualized. Viable cells, in lower left quadrant (Q3), are double negative. Apoptotic cells in
lower right quadrant (Q4) are AnnV⫹ and PI–.
Double-positive cells (AnnV⫹/PI⫹) are in upper
right quadrant (Q2). They are thought to be
necrotic or advanced apoptotic. Cells in last
quadrant (upper left, Q1) are isolated nuclei or
cellular debris. There was significant decrease in
number of viable cells in LPS 14D animal as
compared with control animal. Further, significant increase in apoptotic (Q4) and necrotic
index (Q2) was observed in LPS 14D animal
compared with control animal. Flow cytograms
of C and D, OX42, E and F, 2=,3=-cyclic nucleotide 3=-phosphodiesterase (CNPase), G and H
glial fibrillary acidic protein (GFAP), and I and J,
antineurofilament 200-kd (NF-200) staining of
C, E, G, and I, control and D, F, H, and J, LPS
14D animals. Data were plotted as fluorescence
(FL1) in function of number of counts. Percentage of positive cells in histograms is indicated
by line. There was significant increase in percentage of activated microglia and astrocytes in
LPS 14D compared with control animal. Moreover, proportion of mature oligodendrocytes and
neurons was lower in LPS 14D animal than in
control animal.
Gavilanes. CA induced by intraamniotic LPS resulted in an
interval-dependent increase in CNS injury in the fetal sheep.
Am J Obstet Gynecol 2009.
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FIGURE 3
Apoptosis and necrosis
Differences in percentage of apoptotic, necrotic, and living cells in brain, cerebellum, and spinal cord. Percentage of apoptotic cells in A, brain, B,
cerebellum, and C, spinal cord. Percentage of necrotic cells in D, brain, E, cerebellum, and F, spinal cord. Percentage of living cells in G, brain, H,
cerebellum, and I, spinal cord. Data are represented as mean percentage of positive cells ⫹ SEM. Black bars ⫽ control (n ⫽ 7); gray bars ⫽
lipopolysaccharide (LPS) 2-day (n ⫽ 5); and white bars ⫽ LPS 14-day (n ⫽ 6) groups. Multivariate analysis of variance ⫹ Bonferroni testing; *P
⬍ .05, **P ⬍ .01, and ***P ⬍ .001.
CPU, caudate putamen; HIPPO, hippocampus; hypo, hypothalamus; SN, substantia nigra; WM, white matter.
Gavilanes. CA induced by intraamniotic LPS resulted in an interval-dependent increase in CNS injury in the fetal sheep. Am J Obstet Gynecol 2009.
R ESULTS
Brain and bodyweight
The mean body and brain weights of
the LPS 2-day (n ⫽ 5), LPS 14-day (n ⫽
6), and control (n ⫽ 7) groups are
shown in Figure 1. There were no significant differences in body weight
among the 3 groups (Figure 1, A). The
mean brain weight of both the LPS
2-day (P ⫽ .031) and LPS 14-day (P ⬍
.001) group was significantly lower
than the mean brain weight of the control group (Figure 1, B). A significant
difference between the LPS 2-day and
the LPS 14-day group was shown, with
the last group being most affected (P ⫽
.048 vs control). The percentage brain
of total body weight did not differ significantly between the different groups
(Figure 1, C).
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Apoptotic and necrotic cell death
Figure 2, A and B, shows representative
examples of the histograms of a control
animal and LPS 14-day animal. Percentages of living (AnnexinV– and
PI–), apoptotic (AnnexinV⫹ and PI–),
and necrotic (AnnexinV⫹ and PI⫹)
cells are given in Figure 3. Double-negative (AnnexinV–/PI–) cells are viable
cells (Figure 3, G-I). Values of living
cells were significantly lower in the LPS
14-day group (33-43%) in all CNS areas investigated compared with controls (57-75%). Only the cortex, hippocampus, hypothalamus, and the T11
level of the SC showed a significant decrease in the LPS 2-day group. AnnexinV⫹/PI- cells are considered to be apoptotic cells (Figure 3, A-C). The
percentage of apoptotic cells increased
American Journal of Obstetrics & Gynecology APRIL 2009
significantly in the LPS 14-day group
(28-46%) compared with the saline
group (12-23%) in all regions of interest. In the cortex, hippocampus, hypothalamus, cerebellar cortex, cerebellar
WM, and T11 level of the SC, the percentage of apoptotic cells in the LPS
2-day group was higher than in controls. Double-positive cells (AnnexinV⫹/PI⫹) are considered to undergo
necrosis or advanced apoptosis (Figure
3, D-F). A significant increase in the
necrotic index in the LPS 14-day group
(14-30%) in comparison with the saline group (5-22%) was shown in the
cerebral cortex, CPU, hippocampus
and periventricular WM in the brain,
T11 and L1 level of SC, and in the cerebellar WM. In addition, there was also
a significant increase in the necrotic in-
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FIGURE 4
CNS cell populations
Differences in percentage of microglia, oligodendrocytes, astrocytes, and neurons in brain, spinal cord, and cerebellum. Percentage of microglia in A,
brain, B, cerebellum, and C, spinal cord. Percentage of oligodendrocytes in D, brain, E, cerebellum, and F, spinal cord. Percentage of astrocytes in
G, brain, H, cerebellum, and I, spinal cord. Percentage of neurons in J, brain, K, cerebellum, and L, spinal cord. Data are represented as mean percentage
of positive cells ⫹ SEM. Black bars ⫽ control (n ⫽ 7); gray bars ⫽ lipopolysaccharide (LPS) 2-day (n ⫽ 5); and white bars ⫽ LPS 14-day (n ⫽
6) groups. Multivariate analysis of variance ⫹ Bonferroni testing; *P ⬍ .05, **P ⬍ .01, and ***P ⬍ .001.
CNS, central nervous systems; CPU, caudate putamen; HIPPO, hippocampus; hypo, hypothalamus; SN, substantia nigra; WM, white matter.
Gavilanes. CA induced by intraamniotic LPS resulted in an interval-dependent increase in CNS injury in the fetal sheep. Am J Obstet Gynecol 2009.
dex in the cortex, CPU, hippocampus,
T11 level of the SC, and cerebellar cortex in the LPS 14-day group compared
with the LPS 2-day group. The necrotic
index was not different in the SN, hypothalamus, C1 of the SC, or the cerebellar cortex.
Cell populations
Activated microglia
The representative histograms are
shown in Figure 2, C and D. The number
of activated microglia (OX42⫹) was very
low in the saline group (0.8-1.6%). In almost all regions (CPU, hippocampus,
SN, hypothalamus, L1 level of SC, the
cerebellar cortex, and cerebellar WM),
the percentage of activated microglia was
significantly higher in the LPS 2-day
group (2.5-5%) than in the saline group.
There was an even higher increase in the
proportion of activated microglia in the
LPS 14-day group (3.2-6.8%), affecting
all areas investigated (Figure 4, A-C).
Mature oligodendrocytes
The representative histograms are shown
in Figure 2, E and F. The values of the mature oligodendrocytes observed were similar for the saline (9-52%) and the LPS
2-day (8-57%) groups for all areas investigated (Figure 4, D-F). In the cortex, CPU,
hippocampus, periventricular WM, and
cerebellar WM, the proportion of mature
oligodendrocytes was much lower in the
LPS 14-day group (3-35%) than in the saline group (9-45%). All other regions investigated were not affected in the LPS 14day group.
Reactive astrocytes
The representative plots are shown in
Figure 2, G and H. No significant differences were found in the percentage of astrocytes in the LPS 2-day group
(12-27%) compared with the saline
group (10-22%) (Figure 4, G-I). However, in the LPS 14-day group (14-36%) a
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significant increase in the proportion of
astrocytes was observed in all areas of the
brain and cerebellum. The SC, in contrast, was not affected.
Neurons
No significant differences were found in
the proportion of neurons between the
control (41-82%) and the LPS 2-day (4382%) groups for any of the areas investigated (Figure 4, J-L); representative histogram is in Figure 2, I and J. In the LPS
14-day group (41-73%), however, the
percentages of neurons were significantly lower in the cortex, hippocampus,
and SN compared with the control
group. No differences were detected in
the cerebellum or SC.
There was no influence of sex, singleton/twin, or weight on any of the analyzed variables.
C OMMENT
CA resulted in an interval-dependent increase in CNS injury and different regional
injury patterns. Microglial activation and
apoptotic cell death were increased in all
brain, cerebellum, and SC regions in an interval-dependent manner. In addition,
most brain regions showed reductions in
oligodendrocytes and neurons and an increase in astrocytes, whereas the cerebellum and especially the SC seemed to be less
affected. This study is the first to show a
global inflammatory response to acute CA
in the brain, cerebellum, and SC and a selective vulnerability of the developing
regions.
These changes might be explained by
the state of CNS maturation at that given
gestational age. In the fetal brain, the
WM is predominantly affected by systemic inflammation/infection as highlighted by fetal animal experiments in
rabbits, guinea pigs, mice, rats, and
sheep.19-24 Furthermore, CA is associated with both cerebral palsy and WMD
seen with neonatal imaging.5,7,25 More
recent evidence points toward a relationship between WMD and GM abnormalities.11 This could explain the permanent
neurodevelopmental and intellectual
deficits in ex-preterm infants, which are
not limited to cerebral palsy.26 The use of
this model of CA has several advantages,
making it clinically more relevant than
437.e6
others. First, the CA-associated systemic
inflammation has been previously characterized in this model by Kramer et al.13
Second, the IA LPS strategy avoids the
superimposed hypoxia ischemia of the
IV strategy and the local processes
caused by an intracerebral route.12,27
Third, species with a long gestation duration, such as human beings and sheep,
share several aspects of development and
function. In addition, the current study
not only focused on brain WMD, but it
also analyzed the effect of developmental
inflammation on both the cerebral cortical and subcortical GM structures and
the cerebellar and SC regions. Experimental data combining all these regions
are not available.
The loss of oligodendrocytes, especially in the periventricular and the subcortical WM, has been reported before in
intracerebral, IV, intrauterine, and maternal LPS models in different animal
species.27-31 In addition, a previous
study using a similar ovine intraamniotic
LPS model also demonstrated a decrease
in oligodendrocytes in the areas of extensive focal damage.20 In contrast to our
study, no global effects were previously
found on the density and/or number of
mature oligodendrocytes throughout
the whole brain. Moreover, our study
showed a decreased number of neurons
in the cortex, hippocampus, and SN in
the LPS 14-day group compared with the
control group, whereas neither the cerebellum nor the SC differed from the control animals. GM and/or neuron-specific
damage have not been identified before
in intracerebral or IV LPS models.31 Maternal administration of LPS in rats,
however, led to a loss of tyrosine hydroxylase-positive neurons in the SN of 16month-old offspring.32 In addition, maternal LPS caused the loss of pyramidal
cells in the hippocampus at the age of 8
months.33
Apoptotic cell death was increased in
some of the regions in the LPS 2-day
group (cortex, hippocampus, hypothalamus, cerebellar cortex, cerebellar WM,
and T11) and in all areas studied in the
LPS 14-day group. Necrotic cells were
also higher in most areas of the LPS 14day group compared with the control
group (cortex, CPU, hippocampus,
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periventricular WM, and cerebellar cortex T11 and L1). In a human postmortem study, apoptosis has already been reported as a contributing mechanism for
cell death in infants with WMD.34 Garnier et al35 also found evidence of apoptotic cell death, especially in the
periventricular WM, in a sheep model
for IV LPS. Moreover, an intracervical
LPS injection of pregnant rats led to the
activation of both the intrinsic and the
extrinsic pathway of apoptosis.36 One
possible explanation for LPS-induced
apoptotic cell death could be that LPS
causes the up-regulation of the systemic
production of tumor necrosis factor
(TNF)-␣, activating the TNF-␣-caspase
pathway and an increased production by
microglia and astrocytes in the
brain.13,37,38 Apoptosis has been reported to affect mainly oligodendrocytes
and/or their precursors,34,39 but our
study suggests that neurons are affected
as well.
Microglial activation was increased in
all regions of both LPS groups with the
exception of cerebral cortex and WM,
and the C1 and T11 level of SC in the LPS
2-day group. Increased astrocytic activation was observed in all areas of the brain
and cerebellum in the LPS 14-day group,
whereas the SC was not affected. Similar
increases were found in almost all other
LPS models, in particular in the cerebral
WM.27,29-31 Our study shows for the first
time general microglial activation or astrogliosis throughout the whole brain.
The activation of microglia and astrocytes in the brain may be caused by the
entry of systemic inflammatory cells and
cytokines through a permeable bloodbrain barrier. Activated microglia and
astrocytes respond to an inflammatory
insult, which may affect the WM and,
second, explain the concomitant selective GM compromise. First, neurons
destined for the frontal cerebral cortex
migrate through the developing WM
and form the subplate during late gestation.40 These subplate neurons are the
only neuronal element of developing cerebral WM and persist up to 6 months
postnatally.26 They may guide thalamocortical and cortical axons.26,41 In patients with WMD, apoptotic changes are
seen in subplate neurons, WM, and cor-
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tex.40 Second, oligodendroglial-axonal
interactions are critical for axonal and
neuronal development. Therefore, the
injury to preoligodendrocytes in WMD
could contribute neuronal disruption.26
This GM compromise has been recently
shown by quantitative volumetric magnetic resonance imaging analysis in expreterm infants, where WMD was associated with both a reduced cerebral
cortical and basal ganglia volume and
with a reduced thalamic volume.42-44 In
addition, WM and cortical GM volume
reductions are correlated with working
memory deficits at 2 years of corrected
age and hippocampal volume reductions
are correlated with a memory impairment in ex-preterm infants at 13
years.45,46 Further, qualitative term magnetic resonance imaging analysis with
WMD is positively correlated with superimposed GM abnormalities and
global deficits at 2 years of corrected age
in ex-preterm infants.47
Myelination proceeds from caudal in
the first trimester of gestation to rostral
up until infancy.48 This maturational hierarchy is a plausible explanation for the
lesser general impact of CA on the SC as
compared with the cerebellum and
brain, and the unchanged cerebellar
neuronal counts in LPS-treated animals.
The developing cerebellum is of particular importance because it might not only
influence motor control and posture,
but also the more complex cognitive
processes.49,50
In summary, the CNS inflammatory
impact was universal, because all subjects of a given group were almost
equally affected. The impact was also interval dependent, because damage was
more prominent in the 14-day CA group
than in the 2-day group. Moreover, the
impact showed selective regional differences, meaning that neither astrocytic
and neuronal SC changes nor neuronal
cerebellar changes were found in either
CA group. Whether the differences between groups are caused by: (1) length of
intraamniotic LPS exposure (2- vs
14-day interval); (2) stage of fetal neurodevelopment at the time of LPS exposure
(day 111 of gestation vs day 123 of gestation); or (3) both cannot be differenti-
ated on the basis of the experimental design of this study.
These findings are relevant because
this is the first study showing both global
WM and GM impact of an acute CA in
association with regional differences. f
ACKNOWLEDGMENTS
We thank Dr Vincent Roelfsema and Dr Odette
Besancon for their collaboration and the animal
laboratory team for their assistance during the
experiments. Our special thanks to Prof Marc
De Baets for the generous facilitation of the flow
cytometry equipment.
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